Liver diseases including hepatitis, liver cirrhosis, hepatocellular carcinoma or the like are the most common diseases in Korea, Japan, Taiwan, China, and most of Southeast Asian countries. Among cancer-related deaths, hepatocellular carcinoma is the fourth leading cause of death worldwide (five hundred and five thousand people) (World Health Organization, 1997). In Korea, the incidence of hepatocellular carcinoma ranks third (11.5%) among causes of cancer (Cancer Incidence in Korea, 2002).
To date, a tissue biopsy has been performed or a marker protein of hepatocellular carcinoma such as AFP has been examined for the diagnosis of hepatocellular carcinoma. In addition, several biomarkers have been suggested for the diagnosis, prognosis, or evaluation of treatment efficacy. Among them, AFP and PIVKA-II are the most well-known biomarkers. However, there is still a weak point in their specificity and sensitivity. With recent advances in genomics and proteomics, several candidate proteins and genes as hepatocellular carcinoma markers have been reported. However, the reported genes are mainly used to target the tissue, and there is still no evidence for their secretion to the blood and feasibility of using as serological diagnostic markers. This is because that due to intrinsic properties of biomarkers, most of the studies on tumor markers have focused on their expression differences in the tissue, and their high expression in the tissue does not indicate their availability as diagnostic markers in the urine or serum. Therefore, for convenient diagnosis, it is important to discover tumor markers found in the blood or urine, and there is a need to analyze biomarkers by methods different from previous approaches and develop a diagnostic method for liver diseases including hepatocellular carcinoma.
To solve the above problems, a plurality of tumor markers have been discovered from the blood, tissue, or discharge, but many tumor markers are detected or their expression increased even though cancer development is not observed. Therefore, these markers used for cancer diagnosis are only incidental tools and have not become independent diagnostic tools.
Meanwhile, at the early stage of development, an individual has an immune system which is unique in its ability to distinguish between self and non-self molecules, whereby antigen-antibody reaction (humoral immune response) and cellular immune response are normally induced in response to only foreign antigens exposed to the immune system. However, production of antibodies against self-antigens is observed in certain diseases. In this case, localization of antigen expression is different from that in normal cells, leading to secretion of the intracellular proteins from the cell, or the antigens undergo a conformational change or other abnormal properties are manifested. In regard to cancer, since the 1970s, it has been reported that abnormal growth of cancer cells is accompanied by production of autoantibodies against antigens derived from cancer cells, and these antigens are called as tumor-associated antigens(TAA). Until now, a variety of tumor-associated antigens have been discovered. Among them, HER-2/neu oncoprotein was reported to be a receptor protein located on the cell membrane and induce autoantibodies. A tumor suppressor protein p53 was also reported to induce autoantibodies. In addition, cell proliferation-associated proteins, cyclin B1 and CENP-F (centromere protein F), and onconeurological proteins, Hu and Yo were also known to induce autoantibodies. Taken together, it is inferred that many more autoantibodies against tumor-associated antigens exist, and many trials have attempted to screen tumor-associated autoantibodies in a large scale.
To identify autoantibodies, SEREX (selological analysis of recombinant cDNA expression libraries of human tumors with autologous serum) has been conventionally utilized, by which autoantibodies are detected by selological analysis of protein expression libraries of human tumors with the blood of a cancer patient. However, this method has a limitation in the preparation of diverse expression libraries of tumor-derived proteins. In addition, since final products of the proteins undergo various posttranslational modifications (PTM) after transcription, if it is not considered, protein expression libraries are not sufficient for the detection of autoantigens.
Alternatively, recent advances in the field of proteomics have lead to the identification of autoantibodies. In proteomic technologies, tumor-derived proteins are separated in 2D-PAGE, protein spots are visualized showing a reactivity to the blood plasma of cancer patients as an autoantibody sample, and then the proteins are identified by mass spectrometry. This method is also called SERPA (serological proteome analysis). MAPPing (Multiple affinity protein profiling) is also employed, in which an affinity chromatography resin conjugated with antibodies isolated from the patient's blood is prepared, tumor cell-derived proteins are applied thereto, and bound proteins are identified by mass spectrometry. In another method, a protein chip is manufactured by separation of tumor cell lysate into several thousand fractions, and then the reactivity of a patient's blood thereto is analyzed to detect autoantibodies.
These proteomic technologies have the advantage of directly analyzing the antibody reactivity to tumor cell-derived proteins retaining PTM properties, thereby detecting various autoantibodies, which could not be detected by SEREX. However, these proteomic technologies also have drawbacks.
One is a quantitative problem of antibodies. If the subject to be analyzed is a mixture of two or more, one of them, of which the quantity is greater than those of the others, is dominantly analyzed, and thus the others may be excluded from the analysis. The serum of a patient is a mixture of numerous autoantibodies, and thus the analytical range is determined by differences in their quantity and affinity to antigens, resulting in the failure of analysis of the desired autoantibody. Another problem is that patient-dependency on an autoantibody to be analyzed impairs a systematic analysis on the production of autoantibody in cancer development. In addition, it is very hard to collect an excessive amount of blood from a patient, and therefore, further studies cannot be conducted. The other problem is the conservation of the epitope recognized by an antibody. In accordance with current immunological knowledge, the epitope of an antibody can be divided into two types: a protein sequence-dependent epitope (sequential epitope) and a protein structure-dependent epitope (conformational epitope). In vivo, induction of antibodies against specific antigens is influenced by the physical state of the antigen primarily reacted with the antibody, which indicates that an antigen-antibody reaction occurs in a solution state and the antigen protein maintains its conformation dissolved in the blood. Therefore, upon analysis of antibody-antigen reaction in ex vivo, it is preferably performed in a solution state because their binding is well maintained in the solution. In the above mentioned SERPA, 2D electrophoresis is performed for analysis of the protein mixed solution, in which proteins to be analyzed are denatured using SDS and urea, and the linearized proteins are reacted with antibodies. Thus, if the epitope is a sequential epitope, the antibody-antigen reaction can be detected, but if the epitope is a conformational epitope, the antibody-antigen reaction cannot be detected.